ABSTRACT Clostridioides difficile infection (CDI) is a burden to health care systems worldwide. Gut microbiota dysbiosis associated with CDI has been well accepted. However, contribution of fungal mycobiota to CDI has recently gained research interest. Here, we report the gut mycobiota composition of 149 uniquely well characterized participants from a prospective clinical cohort and evaluate the discriminating ability of gut mycobiota to classify CDI and non-CDI patients. Fecal samples were divided into two groups: (i) CDI (inpatients who had clinically significant diarrhea and positive nucleic acid amplification testing [NAAT] and received subsequent CDI therapy, n = 58) and (ii) non-CDI, which can be further divided into three subgroups: (a) carrier (inpatients with positive stool NAAT but without diarrhea; n = 28); (b) diarrhea (inpatients with negative stool NAAT; n = 31); and (c) control (inpatients with negative stool NAAT and without diarrhea; n = 32). Fecal mycobiota composition was analyzed by internal transcribed spacer 2 (ITS2) sequencing. In comparison to non-CDI patients, CDI patients tend to have gut mycobiota with lower biodiversity, weaker fungi correlations, and weaker correlations between fungi and host immune factors. Notably, 11 genera (Saccharomyces, Penicillium, Aspergillus, Cystobasidium, Cladosporium, and so on) were significantly enriched in non-CDI patients, and Pichia and Suhomyces were enriched in patients with CDI, while 1 two genera, Cystobasidium and Exophiala, had higher abundance in patients with diarrhea compared with CDI (linear discriminant analysis [LDA] > 3.0; P
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